OBJECTIVE: Detect the expressions of the protein tyrosine phosphatase gene (PTEN), phosphorylated protein kinase B (P-AKT) and phosphorylated extracellular signal-regulated kinase (P-ERK) in human middle ear cholesteatoma tissue and its correlation to explore their important role in the mechanism of the formation of cholesteatoma. METHOD: Use immunohistochemical SABC method (SABC immunohistochemical method) to detect the expressions and location of PTEN, P-AKT and P-ERK proteins in 40 cases of middle ear cholesteatoma tissue samples and 15 cases of normal ear skin specimens. Use Western blot to detect the expression levels of PTEN, P-AKT, P-ERK proteins and the internal reference GAPDH in 20 cases of cholesteatoma tissue and 10 cases in nor mal ear skin specimens. RESULT: (1) Immunohistochemistry showed coloring of PTEN both in the nucleus and cytoplasm of cholesteatoma and normal skin . Nuclear PTEN positive expression rates in the cholesteatoma was significantly lower than normal skin, and the difference was significant (P < 0.01); cytoplasm PTEN positive expression rates in the cholesteatoma was significantly lower than normal skin, and the difference was significant (P < 0.01); P-AKT mainly expresses in the cytoplasm of cholesteatoma and normal skin. The positive expression rates in the cholesteatoma was significantly higher than normal skin,and the difference was significant (P < 0.01); the P-ERK mainly colors in cholesteatoma and normal skin cell nucleus. the positive expression rates in the cholesteatoma was significantly higher than normal skin, and the difference was significant (P < 0.01). In cholesteatoma specimens, there was a significantly negative relationship (P < 0.01) between PTEN, respectively, and P-AKT, P-ERK protein. (2) Western blot discovered: the expression of PTEN in cholesteatoma was significantly less than the amount of expression in normal skin; P-AKT and P-ERK expression in cholesteatoma was significantly more than the level in normal skin. CONCLUSION Abnormal expression of PTEN, P-AKT and P-ERK protein in cholesteatoma may be closely related to antiapoptosis and high degree of proliferation in cholesteatoma. Expression of PTEN deletion leads to the weakening of the inhibition. Excessive expression of P-AKT gives rise to cholesteatoma epithelial cell apoptosis inhibited; excessive PERK expression result to cholesteatoma epithelial cell proliferation strengthened.
Apoptosis ; Cell Proliferation ; Cholesteatoma, Middle Ear ; metabolism ; pathology ; Epithelium ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; PTEN Phosphohydrolase ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism

Objective To observe the effects of rehabilitation in the 55 prelingually deaf pediatric cases for two years after cochlear implantation ,factors including cochlear implantation and recovery time ;to compare the re-covery effects in the group of 1 to 3 years old children with the group of 3~5 years old (including the age of 3 years old) who lost their hearing before learning to speak ,and to provide clinical evidence for providing cochlear implant therapy to the prelingually deaf children as early as possible .Methods A total of 55 pediatric relingually deaf cases were included in this study .According to their implantation time and application duration ,they were divided into 2 groups :1 to 3 years old group (32 cases) ,and >3 to 5 years old group (23 cases) respectively .The hearing ,lan-guage and learning abilities on 1 ,3 ,6 ,12 ,18 ,24 months after cochlear implantation were evaluated ,using statisti-cal method to record CAP and SIR scores .Results The rehabilitation effects ,the average ages ,CAP ,speech rec-ognition rates and SIR were increased two years afterwards .The effects of younger age group were more noticeable than that in the older group .The differences between the two groups were statistically significant (P<0 .05) .Av-erage speech recognition rates ,average speech rehabilitation effects in each postoperative period of younger age group were better than those of in older age group ,showing significant differences (P<0 .05);CAPs in the younger age group on 1 ,3 and 12 months after CI surgery were significantly higher than those of in the older group (P value were 0 .001 ,0 .001 and 0 .002 ,respectively) .SIR in the younger age group at the time of 1 ,3 ,12 ,24 months were significantly higher than those of in the older group(P values were 0 .00 ,0 .00 ,0 .00 and 0 .024 ,respectively) . Conclusion Implanted age and recovery time are the key factors that influence the effects of postoperative rehabilita-tion .The younger when the children get cochlear implantation and the longer the recovery time takes during two years after cochlear implantation ,the better the results are .The standardization of domestic assessment for the re-covery effects and the international evaluation method have a certain degree of equivalence .

The human brain-derived neurotrophic factor (hBDNF) gene was cloned by polymerase chain reaction and the recombinant adeno-associated viral vector inserted with hBDNF gene (AAV-hBDNF) was constructed. Cultured rat hippocampal neurons were treated with Abeta(25-35) and serued as the experimental Abeta-induced neuronal damage model (AD model), and the AD model was infected with AAV-hBDNF to explore neuroprotective effects of expression of BDNF. Cell viability was assayed by MTT. The expression of bcl-2 anti-apoptosis protein was detected by immunocytochemical staining. The change of intracellular free Ca ion ([Ca2+]i) was measured by laser scanning confocal microscopy. The results showed that BDNF had protective effects against A-induced neuronal damage. The expression of the bcl-2 anti-apoptosis protein was raised significantly and the balance of [Ca2+]i was maintained in the AAv-hBDNF treatment group as compared with AD model group. These data suggested that recombinant AAV mediated a stable expression of hBDNF in cultured hippocampal neurons and resulted in significant neuron protective effects in AD model. The BDNF may reduce neuron apoptosis through increasing the expression of the bcl-2 anti-apoptosis protein and inhibiting intracellular calcium overload. The viral vector-mediated gene expression of BDNF may pave the way of a novel therapeutic strategy for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Objective:To study the role of DR5 in TRAIL apoptotic signal transduction. Methods:BALB/C mice were immunized with recombinant DR5 that contains the full-length extracellular domain of the human DR5. Anti-DR5 mAb was generated by hybridoma. The level of DR5 expression on Jurkat cell line was examined by flow cytometry. The rates of TRAIL-induced apoptosis and anti-DR5 mAb blocking on Jurkat cells were tested by flow cytometry with TRAIL apoptosis kit.Results:The percentage of DR5 expression on Jurkat cells was 94 83%. TRAIL and anti-TRAIL mAb could kill Jurkat cells on dose-dependent, and the killing rate was 90% in the concentration of 50～100 ng/ml. The killing role of TRAIL could be blocked on Jurkat cells pretreated with anti-DR5 mAb. The average percentage of blocking was 90 49%.Conclusion:DR5 plays a very key role in TRAIL induced apoptosis in Jurkat cells.

Objective To compare the growth of three different cancer cell lines on chick chorioallantoic membrane (CAM), to select the best transplanted cancer cell line for establishing a transplanted tumor model and to observe the biological characteristics.Methods The human lung cancer cell line A549, human tongue cancer cell line TCA8113 and human liver cancer cell line QGY7703 were respectively inoculated into CAM at the 7th day of age.The chick embryo survival rate, tumor survival rate, tumor formation rate and induced angiogenesis were detected and the growth characteristics of the transplanted tumor model were observed.Results Compared with the groups inoculated with A549 cells and QGY7703 cells, the tumor formation rate of TCA8113 cells was the highest (P < 0.05), to be the best cancer cell line for transplanted tumor.The optimal inoculated number of cells was 8.0×106/chick embryo, the optimal growth period of the tumor was 4~8 d, and the best experiment time was 7 d after inoculation.Conclusion The TCA-CAM transplanted tumor model of tongue squamous cell cancer is successfully established for further study of the biological characteristics and mechanisms of tumor growth, angiogenesis, invasion and metastasis, and provide a good experimental animal model for anti-tumor drug screening.

Objective To investigate the effects of estrogen and tamoxifen on the expression of high mobility group box 1(HMGB1) in human breast cancer cell line MCF-7.Methods MCF-7 cells were incubated with 17-beta E2 and tamoxifen at different concentrations for 4 days,respectively,with ethanol as control.Real time RT-PCR and Western blotting were used to detect the expression of HMGB1 at mRNA and protein level after different treatment of the cell line,respectively.Results Compared with ethanol control,HMGB1 mRNA and protein level were significantly decreased when 17-beta-E2 was added at concentrations of 10-6 and 10-4 mol/L(P

The human brain-derived neurotrophic factor (hBDNF) gene was cloned by polymerase chain reaction and the recombinant adeno-associated viral vector inserted with hBDNF gene (AAV-hBDNF) was constructed. Cultured rat hippocampal neurons were treated with Aβ25-35 and serued as the experimental Aβ-induced neuronal damage model (AD model), and the AD model was infected with AAV-hBDNF to explore neuroprotective effects of expression of BDNF. Cell viability was assayed by MTT. The expression of bcl-2 anti-apoptosis protein was detected by immunocytochemical staining. The change of intracellular free Ca ion ([Ca2+]i) was measured by laser scanning confocal microscopy. The results showed that BDNF had protective effects against Aβ-induced neuronal damage. The expression of the bcl-2 anti-apoptosis protein was raised significantly and the balance of [Ca2+]i was maintained in the AAV-hBDNF treatment group as compared with AD model group. These data suggested that recombinant AAV mediated a stable expression of hBDNF in cultured hippocampal neurons and resulted in significant neuron protective effects in AD model. The BDNF may reduce neuron apoptosis through increasing the expression of the bcl-2 anti-apoptosis protein and inhibiting intracellular calcium overload. The viral vector-mediated gene expression of BDNF may pave the way of a novel therapeutic strategy for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Objective To investigate the chemical properties of superficial cardiac neurons. Methods By means of immunohistochemical ABC technique,the study was performed concerning the distribution of calcitonin gene-related peptide(CGRP) and substance P(SP) in the canine main cardiac superficial plexus. Results CGRP-immunoreactive(IR) neurons were found in every plexus,but SP-IR neurons could be observed only in dorsal atria plexus(DAP),inter atria plexus(IAP) and aorta-pulmonary plexus(A-PP).The shape and size of CGRP-IR and SP-IR neurons were similar.The comparative study on atria and ventricles indicated that CGRP-IR and SP-IR neurons in atria were more than those in ventricles.Numerous CGRP-IR,SP-IR nerve fibers could be observed in each fat pats and intermyocardiocytes.These nerve fibers were usually situated near blood vessels,or were attached to vessel wall.Somewhat CGRP-IR and SP-IR nerve fibers were connected with myocardiocytes in some regions.Conclusion The results indicated that actually existed two peptides in canine cardiac superficial plexus.These implied that the regulations of the two kinds of peptidergic neurons to atria and ventricle were different.The two kinds of neurons mentioned above were likely to perform different or similar functions in canine heart.CGRP and SP could possibly modulate the activities of myocardiocytes and vessels of heart directly.

AIM:To investigate the formation of membrane pore in PC 12 cells induced by exogenous adenosine triphosphate ( ATP) and to identify the key molecular targets .METHODS:PC12 cells were treated with different concen-trations of ATP to establish the injury model .The morphological change was observed under an inverted phase -contrast mi-croscope.The viability of the PC12 cells was measured by CCK-8 assay.Fluorescent dye YO-PRO-1 was used to detect the membrane permeability.The expression of P2X7 receptor and pannexin 1 (Panx1) at mRNA and protein levels was as-sessed by real-time PCR and Western blotting .RESULTS:After exposed to ATP (1 mmol/L, 3 mmol/L and 5 mmol/L) for 3 h, the PC12 cells became edematous , and the number of adherent cells decreased gradually in a dose-dependent man-ner .The cell viabilities in 3 mmol/L ATP group and 5 mmol/L ATP group were significantly decreased compared with con-trol group (P<0.05).YO-PRO-1 uptake in the PC12 cells exposed to ATP (0, 1, 3 and 5 mmol/L) for 15 min, 30 min and 60 min increased in a dose-dependent and time-dependent manner .The cell viability increased and the intracellular fluorescence intensity induced by ATP were significantly antagonized in brilliant blue G ( a P2X7 receptor inhibitor ) pre-treatment group (P<0.05), whereas it did not change in carbenoxolone (a Panx1 inhibitor) pretreatment group (P>0. 05).The expression of P2X7 receptor at mRNA and protein levels was significantly increased (P<0.05), but the expres-sion of Panx1 was not changed ( P>0.05) when PC12 cells were exposed to ATP for 3 h.CONCLUSION:Extracellular ATP at high concentration may induce membrane pore formation with the expression and activation of P 2X7 receptor in PC12 cells.

[ ABSTRACT] AIM:To prove the purinergic signaling mechanism of the neuroprotective action of hydrogen sulfide by observing the effects of sodium hydrosulfide ( NaHS) , a donor of hydrogen sulfide, on the cell viability, intracellular Ca2+concentration ( [ Ca2+] i ) and the change of membrane permeability in the PC12 cells injured by adenosine triphos-phate ( ATP) .METHODS: PC12 cells in logarithmic growth phase were randomly divided into 4 groups.In control group, the cells were cultured without ATP treatment.In ATP group, the cells were treated with ATP after cultured for 24 h.In NaHS+ATP group, the cells were incubated with NaHS for 30 min before treated with ATP, and NaHS always exis-ted in the reaction system.In KN-62+ATP group, the cells were pretreated with KN-62 for 30 min, and the other treat-ments were as the same as those in NaHS+ATP group.The cell viability was assessed by MTT assay.The [ Ca2+] i was detected by Fura-2/AM staining.The membrane permeability was observed by staining with fluorescent dye YO-PRO-1. RESULTS:ATP at concentration of 0.3 mmol/L showed no injury effect on the cells.However, the cell viability was dropped gradually in a dose-dependent manner as the ATP at doses of 1, 3, 5 and 10 mmol/L.The decline of cell viability by ATP was obviously reversed by 200 μmol/L of NaHS in the PC12 cells (P<0.05), but exasperated by 800μmol/L of NaHS (P<0.05).At the same time, ATP evoked the increase in [Ca2+]i in a dose-dependent manner, which was inhib-ited by NaHS ( P<0.05) .Furthermore, the YO-PRO-1 uptake induced by ATP in a dose-dependent and time-dependent manner was also reduced by NaHS ( P<0.05) .CONCLUSION:Hydrogen sulfide has protective effect on the PC12 cells injured by ATP.The mechanism may be related to the reverse of the increased [ Ca2+] i and YO-PRO-1 uptake.